Human BMP-7 promoter and method for exploring bone-related substance by using the same

ABSTRACT

The present invention provides a method for exploring low molecular weight compounds which regulate positively or negatively the expression of human BMP-7 with reference to a reporter activity by using 5′ upstream region gene containing the human BMP-7 promoter and an animal cell introduced with the vector that has been connected to an appropriate reporter gene. The low molecular weight compounds and their derivatives obtained by the present method have morphogenetic activity or inhibiting activity for bone and cartilage through the expression of the human BMP-7 and are useful as preventive or therapeutic agents for cartilage and bone diseases. Furthermore, the low molecular weight compounds and their derivatives are useful as therapeutic agents for kidney diseases.

This application is a 371 of PCT/IB99/00733 filed Apr. 22, 1999.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a 5′ upstream region DNA containing apromoter of a human bone morphogenetic protein (hereafter referred to asBMP-7). Further, the present invention relates to a method for exploringa low molecular weight compound positively or negatively which regulatesthe expression of human BMP-7 by using a mass of animal or yeast cellsthat are introduced with a recombinant expression vector harboring a 5′upstream region DNA containing the human BMP-7 promoter integrated intoa suitable reporter gene, and by using a reporter activity as anindicator.

(2) Description of the Related Art

At present, a bone morphogenetic activity has been reported for a bonemorphogenetic factor, BMP, belonging to TGF (transforming growth factor)-β superfamily (Science 150, 893-897, 1965; Science 242, 1528-1534,1988). Known species of BMP are BMP-1 to BMP-14. Among them, the membersfrom BMP-2 to BMP-14 have been known as showing the bone morphogeneticactivity. BMPs ranging from BMP-2 to BMP-14 are considered as effectiveto therapeutic and preventive treatment for various bone dysfunction andbone diseases, however, they exist in very small amount in nature.Therefore, an available large quantity from BMP-2 to BMP-14 used forthese treatments requires production of recombinant protein. Theproduction of the recombinant protein generally is very expensivecompared with a low molecular weight compound. Furthermore, there aremany restrictions as a medical drug in terms of physical properties andadministration methods due to proteinic characteristics. Consideringthese points, a small molecular organic compound having the activityequal to that of the BMP protein, if any, should be a highly promisingmedical drug. The substance obtainable with the exploring methodprovided by the present invention has the activity to induce theexpression of human BMP-7, a bone osteogenesis factor, and also has theefficacy equal to that of human BMP-7, representing very highusefulness. On the contrary, if human BMP-7 is concerned with bone andcartilage hyperplasia, inhibiting the expression may preventosteohyperplasia. The present invention is able to detect the inhibitionof the human BMP-7 expression and provides a method for exploring asubstance to prevent hyperplasia. In addition, it is known that humanBMP-7 has the ability to enhance the differentiation of kidney cells(Proc. Natnl. Acad. Sci., U.S.A., Vol. 93, p. 9021-9026, 1996). Thus,the experimental system provided by the present invention can be appliedto a method for exploring the agent for the treatment of the kidneydisorder.

For such an exploring method, an example has been so far only reportedusing a murine BMP-2 promoter (WO97/15308), and there is no example ofusing the human BMP-7 promoter. In addition, since the materials of theexploring method provided by the present invention are all derived fromhuman sources, it can be expected that discovered substances should showthe effects at clinic practically.

SUMMARY OF THE INVENTION

The present invention provides a 5′ upstream region DNA containing apromoter of human BMP-7. By using 5′ upstream region gene containing thehuman BMP-7 promoter and an animal cell introduced with a recombinantexpression vector that has been connected to an appropriate reportergene, the low molecular weight compounds which regulate positively ornegatively the expression of human BMP-7 can be explored with referenceto a reporter activity. The low molecular weight compounds and theirderivatives have morphogenetic activity and inhibiting activity for boneand cartilage through the expression of human BMP-7 and are effective aspreventive or therapeutic agents for cartilage and bone diseases,remedies for osteometastasis, or therapeutic and preventive agents forexcess osteogenesis. Furthermore, these low molecular weight compoundsand their derivatives are useful as preventive or therapeutic agents forkidney disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exon-intron structure of 10.8 kb 5′ upstream region ofhuman BMP-7 gene and a restriction enzyme map. A net shape shows an exonregion and an open square shows an intron region.

FIG. 2 is a recombinant expression vector (pMSS115) containing a 5′upstream region of human BMP-7 gene. A promoter region (4.4 kb) (baseNo. from 3813 to 8222 shown in SEQ ID NO. 1 of the Sequence Listing,referring from the 2nd XbaI to the 3rd XbaI from 5′ terminal in FIG. 1)was inserted to NheI restriction enzyme site of pGL3-basic.

FIG. 3 is a result of measuring human BMP-7 promoter activity(transiently expression).

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to a DNA whose nucleotide sequence isrepresented by the base sequence No. from 1 to 10877 shown in SEQ ID NO.1 of the Sequence Listing that encodes a human bone morphogeneticprotein-7 promoter region, or a fragment thereof. SEQ ID NO. 1 of theSequence Listing shows the 5′ upstream region sequence of the humanBMP-7 gene.

The present invention relates to a method for preparing the DNA shown inSEQ ID NO. 1 of the Sequence Listing by conducting the steps of:

(1) digestion of a human placenta genomic DNA with a HindIII restrictionenzyme,

(2) isolation by agarose gel electrophoresis,

(3) cloning of the isolated DNA fragment digested with HindIII into alambda phage vector λDASH II treated with the same enzyme,

(4) packaging of said vector into the phage,

(5) establishment of genomic DNA library by infecting Escherichia coliwith the phage,

(6) screening by PCR, and

(7) subcloning into a plasmid vector.

The plasmid vector used herewith is not restricted and can be used amongones commercialized. A pUC18 vector can be a preferable example.

The present invention relates to a recombinant expression vectorcharacterized by integration of the full length or a part of DNA shownin SEQ ID NO. 1 of the Sequence Listing into a reporter gene. In detail,the recombinant expression vector is constructed to locate a suitableregion of 5′ upstream region of the human BMP-7 gene, that isrepresented by SEQ ID NO. 1 of the Sequence Listing, in front of areporter gene. The reporter gene such as luciferase or β-galactosidasegene shows an expressing status on behalf an original product. Thevector as the original for the recombination expression vector is notspecially restricted to allow to use a plasmid vector commercialized.The present invention used pGL3-basic as a preferable example. The useof pGL3-basic yielded pMSS115 (9.2 kb) that is a recombinationexpression vector containing the human BMP-7 promoter and a luciferasegene. The present invention assigned it to the recombination expressionvector. It is necessary to introduce the vector to mammalian cells,preferably a human osteoblast-like cells, such as SaOS-2 cells, with aliposome. The animal cells stably transfected with the recombinantexpression vector are selected by using a resistance marker.

The present invention relates to a method for exploring a bone-relatedsubstance, characterized by using the recombinant expression vectorcharacterized by integration of the full length or a part of DNA shownin SEQ ID NO. 1 of the Sequence Listing into a reporter gene. It relatesto the method for exploring a bone-related substance wherein thebone-related substance is osteogenesis inducing substance or abone-related substance wherein a bone-related substance is osteogenesisinhibiting substance. A low molecular weight compound which induces orinhibits the expression of human BMP-7 can be obtained by isolating thepromoter which regulates the expression of the gene, by connecting it toa suitable reporter gene and by introducing the gene structure to asuitable mammal cell to make an exploring system. The substance whichregulates the expression of human BMP-7 in the exploring system works onthe promoter to increase or decrease the expression level of thereporter gene. Therefore, a simple and easy measurement of the reporteractivity makes an exploration of the aimed substance possible.

The animal cell transfected with said vector can be used for a methodfor screening a chemical compound library by high throughput screening(Nature, Vol. 384, Suppl., p. 14-16, (1996) and exploring an activesubstance from natural substances. The substance which increases ordecreases an activity is searched by treating the cell with a substancefor an appropriate time period and thereafter measuring the reporteractivity. The substance obtained hereby can regulate the expression byworking directly on a transcription factor or indirectly on the promoterof human BMP-7 through regulating a signal transduction system.Therefore, these compounds are effective as a therapeutic agent forosteocartilaginous diseases, cancer metastasis to bone, orosteohyperplasia.

Furthermore, these compounds are useful as a therapeutic agent forkidney disorders.

The substance obtained by the present invention has bone or cartilagemorphogenetic activity and is effective as an agent for therapeutic andpreventive treatment in the fields of orthopedic surgery (fracture,osteoarthritis such as joint osteoarthritis and hip jointosteoarthritis, arthrosteitis, damage of cartilage such as damage ofmeniscus, regeneration of bone and cartilage deficit caused by injuryand tumor dissection, bone reconstruction such as spinal fusion andvertebral canal enlargement, and congenital cartilage and bone diseasessuch as dysoteogenesis and achondroplasia), or dental fields (bonereconstruction such as palatoschisis, mandible reconstruction, andresidual ridge construction), and osteoporosis. Moreover, the substanceof the present invention can be used for bone graft in aestheticsurgery. These therapeutic treatments are effective to therapies in thefields of veterinary surgery. On the other hand, the present inventioncan provide a substance to inhibit bone or cartilage morphogenesis. Inthis case, the substance is applied as an agent for prevention andtherapy of bone and cartilarge hyperplasia.

In addition, the present invention can provide a substance with abilityto enhance the differentiation of kidney cells and it can be applied toan agent for the treatment and prevention of the kidney disorder.

EXAMPLES

This invention shall be more illustratively explained by way of thefollowing Examples. The following Examples are to be considered in allrespects as illustrative and not restrictive.

Example 1

Isolation of 5′ Upstream Region of Human BMP-7 Gene

A human placenta genomic DNA (a product of CloneTech) was digested byusing various kinds of restriction enzymes (BamHI, BglII, EcoRI,HindIII, PstI, SacI, SalI, Smal, SphI, and XbaI), separated by agarosegel electrophoresis, transferred to a nylon membrane, and subjected tothe Southern hybridization using BMP-7 cDNA (EMBO J. 9: 2085-2093, 1990)as a probe. As the result, it was found that digestion by therestriction enzyme HindIII among restriction enzymes used yielded a DNAfragment of ca. 11 kb containing the longest human BMP-7 gene. Then, ahuman placenta genomic DNA was digested by the restriction enzymeHindIII and separated by agarose gel electrophoresis to extract a DNAfragment of ca. 11 kb from the agarose gel. The DNA fragment obtainedwas cloned to lambda phage vector λDASH II (Stratagene Ltd. made)digested by the restriction enzyme HindIII. The vector was in vitropackaged by Gigapack III XL Extract (Stratagene Ltd. made), infected toEscherichia coli XL1-Blue MRA (Stratagene Ltd. made) to make a genomicDNA library. The library was divided into pools. Each pool was amplifiedby a screening (Nucleic Acids Res. 21: 2627-2631, 1993) using PCR;namely, the PCR method by using PCR primers (SEQ ID NO. 2 and SEQ ID NO.3 of the Sequence Listing) corresponding to translation region to selectthe objective pool, to obtain finally 5′ upstream region (10.8 kb) ofhuman BMP-7 gene. In addition, the 5′ upstream 10.8 kb fragment wassubcloned to a pUC18 vector (a product of Amersham Pharmacia Biotech).The vector was named E. coli pKOT 314. The E. coli pKOT 314 wasdeposited in National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Ministry of InternationalTrade and Industry 1-3, Higashi 1-chome, Tsukuba-shi Ibaraki-ken305-8566 Japan, in Mar. 30, 1998 with depository number FERM P-16737 andtransferred to the International Depository Authority under BudapestTreaty on Feb. 17, 1999 (Deposit No. FERM BP-6651).

Example 2

Determination of DNA Sequence of 5′ Upstream Region of Human BMP-7 Gene

The sequence of 5′ upstream region of human BMP-7 gene obtained wasdetermined by Amersham Pharmacia Biotech's ALF DNA Sequencer accordingto the method of Sanger et al. (Proc. Natl Acad. Sci. USA 74: 5463-5467.1977). The sequence thus analyzed is described in SEQ ID NO. 1 of theSequence Listing. The base sequence No. from 5557 to 10780 of SEQ ID NO.1 was already been reported (EMBO J. 9: 2085-2093, 1990). However, thereare many differences from the sequence of this invention.

Example 3

Construction of a Recombinant Expression Vector Containing the HumanBMP-7 Promoter and a Luciferase Reporter Gene

As shown in FIG. 1, the promoter of human BMP-7 exists in the upstreamof exon 1. Then, a region of 4.4 kb containing a promoter from XbaI ofthe second position to XbaI of the third position from the 5′ terminaldescribed in FIG. 1 were—to align in the upstream of the reportergene-inserted in the restriction enzyme site, NheI, of a luciferasereporter vector pGL3-basic (a product of Pro Mega Ltd.) to construct arecombinant expression vector pMSS115 (9.2 kb). This is presented inFIG. 2.

Example 4

Measurement of the Activity of the Human BMP-7 Promoter (Introduction ofa Recombinant Expression Vector to a Human Cell and TransientExpression)

In order to express transiently the human BMP-7 recombinant expressionvector, said recombinant expression vector (pMSS115) was mixed with avector, pRL-SV40 (a product by Pro Mega Co.) containing sea pansyluciferase gene as an internal control for measurement of efficiency ofgene introduction in equal quantity. Then, cationic liposomelipofectamine (a product of Lifetech Oriental Co.) was added to themixture solution to add to human osteosarcoma cells HOS, MG63, andSaOS-2 for transfection. Fire fly luciferase activity and sea pansyluciferase activity were measured by Pikka Gene Dual Kit (a product ofToyo Ink Co.). The result is presented in FIG. 3. The promoter activitywas expressed as a ratio of fire fly luciferase activity to sea pansyluciferase activity. From the result, it has been known that the DNA ofSEQ ID NO. 1 of the Sequence Listing has a promoter activity.

Example 5

Introduction of the Recombinant Expression Vector to a Human Cell andStabilized Expression

In order to express the human BMP-7 recombinant expression vectorstably, said vector was mixed with a vector pPUR (a product of CloneTechLtd.) containing puromycin resistant gene in the proportion of 10:1 andalso mixed with cationic liposome lipofectamine (a product of LifetechOriental Co.) to add to a human osteosarcoma cell HOS for transfection.The cells to which the aimed gene had been introduced were selected froma culture medium containing puromycin (a product of Sigma Ltd.).

Example 6

Screening of Active Low Molecular Weight Compound

The established cells selected were inoculated in a 96-well plate,treated with substances of various chemical compound libraries for 1-3days, dissolved with a cytolytic agent (a product of Pro Mega Ltd.), andmeasured for enzyme activity by employing a luciferase assay kit (aproduct of Pro Mega Ltd.). By such processes, various substancesinducing or inhibiting the expression of human BMP-7 are obtained.

Sequence Listing Free Text

<210> 1

<223> Human BMP-7 5′ upstream gene sequence including the exon 1regions.

<210> 2

<223> Sense PCR primer for cloning 5′ upstream human BMP-7 gene sequencecorresponding to the exon 1 region.

<210> 3

<223> Reverse PCR primer for cloning 5′ upstream human BMP-7 genesequence corresponding to the exon 1 region.

3 1 10877 DNA HUMAN misc_feature (1)..(10877) Human BMP-7 5′ upstreamgene sequence including the exon 1 regions. 1 aagcttggac atcagatagctgtgtcattc atgctaactc ttattagttg ttgcatcata 60 tgtaagatga gctcaaatctgttaatcttc ccatacgatc ttttcacatg agttgggact 120 aacctgttat atttagagtaatggtttacc aatggggagg gaataaagat aaaactcgaa 180 gtaaaagaaa gaatacaattttttaaatga ggagaaacac agtgaaggag aggagagaag 240 gaagaaaaca acagagatggagagaagcac gacgtggggt ggtgaggagt tcccaggact 300 ctgggagcga ggagcccagtctaatccctg attcactgta agactttggg cccagcagtt 360 tctctttggg tgtcaatttctctatcttca agatgaagga attgaaccac gcttctgcag 420 gacttttgtt gtaactgactggaaactccg tacacaatgg cttaaactga acagtcctgg 480 gtggggctgg cttcagggatagtgggctcc aggggctcac tgatgctgtc agtctcctgc 540 cttttcttgt tttactgtcttgtggcttct ctcacagggc aggctctccc tgaagggagg 600 gaagatgaca gccagcagcagcccctgacc tagcaacccc agtgggagga gagacccctt 660 taccggtagt tctagcaaaagttctaaaag ttgaatctca ctggcctgga ttaggtcaca 720 tgactgtcac tgaacccatcgcatgggcat ctctggttga ccaggctggg tcatgtgccc 780 agtctgaatt ttagagaagaggatagttca acctgaaata cagggactga tggtgaagaa 840 ggggcatttc tactgaggaaaacagaggaa atcttggcca ggcacagtgg cttatgcctg 900 taatcccagc actttgagaggccaagatgg atggattgtt tgaggcccgg agttcaagac 960 cagtctggcc agcgtggtgtaaccctgtct tctactaaaa atacaaaaat tagccaggca 1020 tggtggtgca cacctgtaattccagctact cgggggggct gaggagggag aattgcttga 1080 aaccgggagg cagaggttacagtgagctga gattgtgcca ttgcactcca gcctgggtga 1140 cagagggaga ctctgtctaaaaaaaaaaag aaaagaaaag aaaacagagg aaatatttgc 1200 agaagaagga gaaaaggcaggggtctcaaa ttaggtgagc ttccaaagtg ttttctgtgg 1260 aaccctggga gcagagaaaatggtagtgaa aagaaacagg cctggggact ctgggcccct 1320 gctgggatca cctgataacaggcagggtgt ccagtccgat ttgaacttca gataaacaac 1380 gagtaaattt cagtatgagtatatataata catatactcc aatataatgg gatatagtca 1440 tactaaaatt cattcattgtttatctgaaa ttcaaattga actggacacc tatttttttt 1500 tttgcaaaat gtggtaaccctagtccctcc cctgctctat gtaaccaaag aagctctgtt 1560 tctgttttct gtgtcgagcatgtgtgaaaa actccttgac caaaagtttt agcgctgcac 1620 aaaagagaag gcctgtatccttttaactga catgattgcc atgccctctc cagctcccca 1680 gtgctgtatc cagcagggcttctggctgca ggcaacagaa accagcttgg gcaggctggt 1740 gcggaaaagg agaccactagaggatcttgg gggctcacag aacccctggg aaggccagag 1800 aaccaggctt gggggctctctagttgggac acggccccag atcactctgc agaatagtct 1860 gggcctgcac tgatagcttctggggtgggt gcctctgact ggctgaaact tggtcacctg 1920 tcagggctct ggctgcaaagcaggctagac agtgcctctc tggatattca ccagatgggg 1980 catcctcaac cataagagagggctcagatc tagctgggag gagggagatg ctgaaaagca 2040 ggagcaatga acactcactcctgctcttgc agatggaatt ctggtttttg atgtttctcg 2100 ggagaggggg agtttatcagatcagtcacc agggccaaac agcagagttt tgctgggaag 2160 ggactctggg gaaggttagggtcagatttt gcctaaaggg gcagacagtt ccagcctttt 2220 caaccactgg ctcagtagccctcgaatcat gaattcaatg gagctgtctt gaaatgcacc 2280 atccttggtg gtcctttggtttgggtgatg ggttggggga ggctgggcac atcctccaga 2340 cacagcagag ccctttatgaggccaggggc aaaccagatt catcgaatct gactggtaga 2400 tgtgtcttct ttggtctaataaccaaaacg agttgaatta gttgccaaca gataaacatt 2460 gagagatgtc acattcaaactcaagtttct agcttctttg gaaaatcaga actttctggt 2520 gatgttggaa tcaaatttctggatggcatc acccatgggc atgtggtttc cagttcaccc 2580 cagtctgcac cctccttatttatccaggcc agtgtctcct gccacctgtc accatgtctg 2640 tgctcatgat tgccttccattaaattaaga ggaagagaaa tattccttga gcttcatgtt 2700 gcatctctgt atttatgttacctggttggt gcccgttggc ttttgagtgt atgacttctg 2760 caataaagga ggaattgaagaagggctcag aggtatgttc ctcaaacact gatagggcat 2820 ctactgtgtg caaggcctccatgggtactg ggaggtccct acaggactaa gacaaaccct 2880 gtgttttggt gtacctcaatgtttaaggac ttaagtcctg gaacaagata gccggcgtca 2940 gaattctgat tttaccattcactcaccatt ggtgtggctt tgggcaggtt ccctcaagta 3000 tttgagcctc aatttcctcatctgtaaaat ggggataata ataataaaaa tcatacaagg 3060 tagttgggag aattaatgagttaattttaa aagacacctg gaatcataga tattgaagtt 3120 ttaatttgaa gccccaatttgtcagctata acttcagaaa tctagagcct cgatcgtcaa 3180 catacagcct gaagacgacagcctccgagc cccccaggag ctagttagaa gtgcagaatc 3240 tcagctcact cagactgactgtgggaatct atgttctaac agacccccag gggattcaca 3300 cgcacagttg ggttggagaagtgttgcttt aggttaaagc aaggaaggaa ggggctgctc 3360 ccaggtcact gaacgtgcactgatgcttaa aggcctgggc tgtgtagaca gatgtggctt 3420 tggatcctgg ctccattacagattagattg gctgtctgac tctggacaag gtgctctcgg 3480 gatgttggtc ctcctctgcaatctgaggag cagaactagc ctctccatgc agtggggagt 3540 tgcgggtgat gtgggcttgggccgtgcagg cctagcaggt gcccagcaga agcaagtgct 3600 cccaacaggt gacaggtcggccgctcccct gtcacgtttt ggaaggagga aagggctacc 3660 tctagtgctg aaccgaggatagttagtgct caaaaccata ccagatttcc tccaatcaag 3720 gaagaaataa cagccctgataataataaac aaaaccactg cttcccccta gctggcttcc 3780 agatcctaga aatcctgcatggtatcagct tctctagagt gtgtgtgtcg tctgtatttt 3840 ccatttggaa ctgtggccactgccatgtgt acttaagact gatggaagac gtttaatctc 3900 ttattctgct gcttttaaagttgtgcaaag aaaatctcag agtgggcaag cgtgaggagt 3960 ccaagcctcc cgtgcaatgagagatctgcg tgggaaacaa aatttcacca caggtgtgct 4020 ctaataattt cctctgcagggtccacctct gcacacaacg attttcaagc ctgtgcagga 4080 ggcagctctt ggccctctattccttgttgg ggttggaggt ccaggtcatc tggcctttgc 4140 caggtggtgt ggacagagagagagcagccc actggcttcc ttggctgtgc tccccaggga 4200 gcttccaggc cagctgagcctcctctaggg cagatggatg aagatgaggt ccccacctcg 4260 ctgaacaatg tgtgtgtcagcagaattctt ttcttctttt tactgcctta gagagacatg 4320 ctgacttggt caaaatcacttcagcaagat gtgactgatg atgtaggaaa tgaaaactcc 4380 tggcccagca ctgggaggccagcaggcgag gggcgcggac actgggaccg ggccacccga 4440 cacaataaca ccccttacaattccaggccg tctttcatcc aggcaaaggc ggccccaggt 4500 gtggagcaga ctggggcagatcagattacg gtcctggagc ccagtgctgg gtgccctggc 4560 caggggaacc acaactgggggtgtgtgggt tgggggctag ctgctgggga aatgagatag 4620 gacccaggga agtccctgggccccagctgg cctgcagggg gcctaggcac aatgtgagga 4680 ttggaatgca gtgatgaccttttcatctct catgttccct tccacttcac tctctctttt 4740 gctctggtac ttttgctcttgctctcagag tcaggataca tctgcttttc tctgcttggc 4800 aaaaagcaga ccatcatgaaaagttttgtt gtaatttgaa actcaggagg ctttcctgtt 4860 accatatttt ccttttcatccagatggtaa cacaataaca tttatcaagg gttttctctg 4920 tgttccaggc actgtgcaaagccctttcca tgaattaagt tctgcaacct cacagcgatc 4980 ccagaagaca ggcgctatcagtccccccat tttacagatg ggggaactga ggtgtagaga 5040 ggttaagtcc cttgcccatggtgcacagct ggaagagaca gagctggagt gtgaatgcgg 5100 ctgggcaggc tccagtgcccaggctacctc ctccacacaa gacttgccct cggcaatctc 5160 aaagcctttt ctggtggtgggctcagctcc caaactggca tcggatgcac tcccagccag 5220 atatttcttg cttgccggttttcattcatt cattcattca ttcattcatt cattcattcg 5280 gcattcactg agggcctggtgcagtcttgg ggatgcctct cggggaggaa acagggaaaa 5340 gaaagacccc cccaccaagcatggatcaca gaaaagataa ggctaaatgg gggtttgtgg 5400 gacttcagag gaaaccttatctcttgaggt cttggatatg aagagcatgt tgtctcttcc 5460 tcttgcatga gaaaagatggcgtctcagag gaagggttgc tggggtgagg gatctgggag 5520 atgccttagc ttggcgcctgcacagtcagc cctcagtcaa ccggtctctt taggttttgg 5580 ctgtgcttat tactattcattcaacaggta ctaattgagc acctgctgtg tgccaggctc 5640 agaataggct caggtgagatgcacaaagaa gggtaaacta gaatccttgc ttagacactg 5700 acggatcagt tgtttcatatgtaaattgta gcaccaagac ctgctgcccc tgcccccagc 5760 ctcacctgct tgtgaagatccctccaaaag atttgagagt agataaaaag cagagactac 5820 tactgaagaa cagggctgctttggctcctt attatttcag actttggaag aaaatgacct 5880 cctttttctc tactggcactggaggtggca tagctgtccc tagcaagcca gcgctggagg 5940 gcgtgtgcag ggctggggaccgagcctggt ttctgttccc tgctctgcag gctcaagcac 6000 ttgctgttcc tccacctgggatgcctttcc ctggaaaagc ctgtctcttt cttgtctttc 6060 aggactcagg tcagtggcatctcctccaaa aactcccctt cccaccctcc atcacctcac 6120 cctgtttatc tgcgcccccgcccccactgc ctgtcactta ttgcaggctg aagtgaccca 6180 ggctctccag ttgtacactctcagatggac cctggacgac tgtggcactc ctgcaatttc 6240 cccagtctcc ctggggtaggattcctgctt gccaggatgc ccacctttcc ttctccctcc 6300 tgcatgtcct cctctgcctggcttctgaat tgttcccaga gagagtgata gacaagatct 6360 gcctctcctt cagtccctgaatcttattta aggctcttgc tttgcttccc tggcctggag 6420 gcggctcctt gatggagtctgccatgtggg ttcgctcatg gccatgtctt cctgcccagc 6480 atggtgcttg gccctgggactggccacata atatctgggc caggtgcaaa attagtacgg 6540 ggcagggggt actttgttcataggtgattc agaaccacat atggtgacct cagagtagga 6600 aaccaagtgt ggggcccttaagagctgggg ggccctgtac gactgtccag gttgcaggcc 6660 ccacagctcg cctcctgatatcctgtgctc catgcttgtc tgttgaagga aggagtgaat 6720 ggatgaagag caggtggtgggggtggtttg agggccttgc tggtgggtgg gtagaggccc 6780 ctccctggca tggggctcaagacctgttcc atcccacagc ctggggcctg tgtgtaaatg 6840 gccaggacct gcaggctggcatttttctgc tccttgcctg cctctggcct cccctttctc 6900 cacccatgtg gcccctcaggttgccatcta gtccaaaagt ccccaaggga gacccagagg 6960 gccacttggc caaactacttctgctccaga aaactgtaga agaccataat tctcttcccc 7020 agctctcctg ctccaggaaggacagcccca aagtgaggct tagccagagc ccctcccaga 7080 caagcgcccc cgcttccccaacctcagccc ttcccagttc atcccaaagg ccctctgggg 7140 acccactctc tcacccagccccaggagggg aaggagacag gatgaacttt taccccgctg 7200 ccctcactgc cactctgggtgcagtaattc ccttgagatc ccacaccggc agagggaccg 7260 gtgggttctg agtggtctggggactccctg tgacagcgtg catggctcgg tattgattga 7320 gggatgaatg gatgaggagagacaggagag gaggccgatg gggaggtctc aggcacagac 7380 ccttggaggg gaagaggatgtgaagaccag cggctggctc cccaggcact gccacgagga 7440 gggctgatgg gaagccctagtggtggggct ggggtgtctg gtctcaggct gaggggtggc 7500 tggaaagata cagggccccgaagaggagga ggtgggaaga acccccccag ctcacacgca 7560 gttcacttat tcactcaacaaatcgtgact gcgcacgtac agtggctacc aggcgctggg 7620 ttcaaggcac tgcgggtaccagaggtgcgg agaagatcgc tgatccgggc cccagtgctc 7680 tgggtgtcta gcgggggtaagaaggcaata aagaaggcac ggagtaactc aaacagcaat 7740 tccagacagc aagagaaactacaggaaaga aaacaaacgt gcgaggggcg aggcgaggaa 7800 acaacctcag cttggcaggtcttggaggtc tctgggagga gaaagcagcg tctgatgggg 7860 gcgggaggtg gtgagtggggagaggtccag gcggagggaa tggcgagcgc agagacaggc 7920 tggcaacggc ttcagggaggcgcggagggg tcagcgtggc tggcttaaaa ggatacatgg 7980 gactgagggg caagaccggctcaagggtca ccgcttccag gaagccttct atttccgcgc 8040 caacctcggc gctcccccaacttttcccac cgcggtccgc agcccacccg tcctgctcgg 8100 gccgccttcc tggtccggaccgcgagtgcc gagagggcag ggccggctcc gattcctcca 8160 gccgcatccc cgcgacgtcccgccaggctc taggcacccc gtgggcactc agtaaacatt 8220 tgtcgagcgc tctagagggaatgaatgaac ccactgggca cagctggggg gagggcgggg 8280 ccgagggcag gtgggaggccgccggcgcgg gaggggcccc tcgaagcccg tcctcctcct 8340 cctcctcctc cgcccaggccccagcgcgta ccactctggc gctcccgagg cggcctcttg 8400 tgcgatccag ggcgcacaaggctgggagag cgccccgggg cccctgctat ccgcgccgga 8460 gttggaagag ggtgggttgccgccgcccga gggcgagagc gccagaggag cgggaagaag 8520 gagcgctcgc ccgcccgcctgcctcctcgc tgcctccccg gcgttggctc tctggactcc 8580 taggcttgct ggctgctcctcccacccgcg cccgcctcct cactcgcctt ttcgttcgcc 8640 ggggctgctt tccaagccctgcggtgcgcc cgggcgagtg cggggcgagg ggcccggggc 8700 cagcaccgag cagggggcgggggtccgggc agagcgcggc cggccgggga ggggccatgt 8760 ctggcgcggg cgcagcggggcccgtctgca gcaagtgacc gacggccggg acggccgcct 8820 gccccctctg ccacctggggcggtgcgggc ccggagcccg gagcccgggt agcgcgtaga 8880 gccggcgcga tgcacgtgcgctcactgcga gctgcggcgc cgcacagctt cgtggcgctc 8940 tgggcacccc tgttcctgctgcgctccgcc ctggccgact tcagcctgga caacgaggtg 9000 cactcgagct tcatccaccggcgcctccgc agccaggagc ggcgggagat gcagcgcgag 9060 atcctctcca ttttgggcttgccccaccgc ccgcgcccgc acctccaggg caagcacaac 9120 tcggcaccca tgttcatgctggacctgtac aacgccatgg cggtggagga gggcggcggg 9180 cccggcggcc agggcttctcctacccctac aaggccgtct tcagtaccca gggcccccct 9240 ctggccagcc tgcaagatagccatttcctc accgacgccg acatggtcat gagcttcgtc 9300 aacctcggtg agtaagggcaggcgagggta cgcgtctcct ttcgggggca ctttgagact 9360 gggagggagg gagccgcttcttctatgcag cccgcccagc tttccgctcc tggctgaaat 9420 cgcagtgcct gcccgagggtctcccaccca cagccctatg actcccaagc tgtgtgcgcc 9480 cccaggtcgg gcgcgctgggttcggtgagc ctgtaggggt tactgggaag gagggatcct 9540 ccgaagtccc ctccatgttacgccgccggc cgcatctctg gggctggagg caagggccgt 9600 tcaaagcgcg gggctcggtcatgtgagctg tcccgggccg gcgcggctcg cgtaacctgg 9660 atgtaaaggg cccttcccggcgaggctgcc ttgccgccct tcctgggccc ctctcagccc 9720 tgcctggccc tggcatcgcggccgtcgcac ccccttaccc tccctgtcaa gccctacctg 9780 tcccctcgtg gtgcgcccgccttagcgtac cgcgcgctcc gagcgcttgg ggcccctctc 9840 cgggccgccg gatgccccattctctcttgg ctggagctgg ggaagaaacg gtgccattgc 9900 taattttctt tgttttctttctttgtttat tttttttctt ttttcttttt ttttcttttc 9960 ttttcttttc ttttttttttttttttgaga cggagttcac tcttgtcgcc cagtctggag 10020 tgcaatggcg cgatctctgctcaccgcaac ctctgcctcc cgggttcaag cgattctcgt 10080 gcctcagcct cccgagtagctgggattaca gcatgcgcca ccatgcctgg ctaattttgt 10140 atttttagta gagacagggtttctccatgt taggcaggct ggtctcgaac tcccgatctc 10200 aggtgatcct cccgcctcagcctcccaaag tggtgctggg attacaggcg tgagccactg 10260 tgccctgccg ctagtcttctattttaagta tttagtggta ggtcccgggc cggcagaatc 10320 tattttcagc atttaccacgtgtggcgcgc aaaccacagg ttttggcgat tgggttgcgc 10380 gggatctcag actgacgcgcgggggcggct gggggtcccg gtttccgact ggagccgcga 10440 cgaccccggc gacgcgagcctggggctgca gcgagggccg gggagctccc cctccatatg 10500 tgcgcgcaca ttctccagacttgctcaaac taaccccccg cggcgccagc gcgctgcggg 10560 actgatgatc aaatatttggtttccgagat aacacacccc gatagcgctg tttcctgagc 10620 cgctttcatt ctacttgtgtaacttgctgc gaaaacccga accaagtcaa gacagcaaac 10680 tcaccccacg ggcgctgtgtcaacatggaa ataatgatac tgaagcccca cgctgggcac 10740 ctggggcgtg gactgggggcgcgggggaag cgcagatccg ccttcatgct tcccccctcc 10800 tgataaggtc cctggagttcccgggaggcc attgtctgta cttaataata actaaatcca 10860 actagtgaac caagctt10877 2 30 DNA HUMAN misc_feature (1)..(30) Sense PCR primer for cloning5′ upstream human BMP-7 gene sequence corresponding to the exon 1region. 2 gggcgcagcg gggcccgtct gcagcaagtg 30 3 30 DNA HUMANmisc_feature Complement((1)..(30)) Reverse PCR primer for cloning 5′upstream human BMP-7 gene sequence corresponding to the exon 1 region. 3agaggatctc gcgctgcatc tcccgccgct 30

What is claimed is:
 1. An isolated nucleic acid corresponding to SEQ IDNo: 1 that encodes a human bone morphogenetic protein-7 promoter region.2. A method for preparing isolated nucleic acid corresponding to SEQ IDNo: 1 comprising (1) digesting a human placenta genomic DNA with aHindIII restriction enzyme, (2) isolating by agarose gel electrophoresisa DNA fragment, (3) cloning of the isolated DNA fragment digested withHindIII into a lambda phage vector λDASH II treated with the sameenzyme, (4) inserting the said vector into the phage, (5) establishing agenomic DNA library by infecting Escherichia coli with the phage, (6)screening by PCR, and (7) subcloning into a plasmid vector.
 3. Arecombinant expression vector comprising the human bone morphogenicprotein-7 promoter region of claim 1 operably linked and 5′ to areporter gene selected from the group consisting of luciferase andbeta-galactosidase.
 4. A method for identifying candidate compounds forinhibiting or inducing osteogenesis, which candidate compounds increaseor decrease human bone morphogenic protein-7 promoter activity,comprising introducing the recombinant expression vector of claim 3 intoa mammalian cell in vitro and measuring a change in reporter geneexpression in the presence of said candidate compounds.